One approach to treating individuals infected with HIV-1 is to administer to such individuals compounds that directly intervene in and interfere with the machinery by which HIV-1 replicates itself within human cells. Lentiviruses such as HIV-1 encode a number of accessory genes in addition to the structural gag, pol, and env genes that are expressed by all replication-competent retroviruses. One of these accessory genes, vif (viral infectivity factor), is expressed by all known lentiviruses except equine infectious anemia virus. Vif protein of HIV-1 is a highly basic, 23-kDa protein composed of 192 amino acids. Sequence analysis of viral DNA from HIV-1-infected-individuals has revealed that the open reading frame of Vif remains intact. (Sova, P., et al., J. Virol. 96:2557-2564, 1995; Wieland, U., et al., Virology 203:43-51, 1994; Wieland, U., et al., J. Gen. Virol. 78:393-400, 1997). Deletion of the vif gene dramatically decreases the replication of simian immunodeficiency virus (SIV) in macaques and HIV-1 replication in SCID-hu mice (Aldrovandi, G. M. & Zack, J. A., J. Virol. 70:1505-1511, 1996; Desrosiers, R. C., et al., J. Virol. 72:1431-1437, 1998), indicating that the vif gene is essential for the pathogenic replication of lentiviruses in vivo.
In cell culture systems, vif-deficient (vif−) HIV-1 is incapable of establishing infection in certain cells, such as H9 T cells, peripheral blood mononuclear cells, and monocyte-derived macrophages. This has led to classification of these cells as nonpermissive. However, in some cells, such as C8166, Jurkat, SupT1, and HeLa-T4 cells, the vif gene is not required; these cells have been classified as permissive. (Gabuzda, D. H., et al., J. Virol. 66(11):6489-95, 1992; von Schwedler, U., et al., J. Virol. 67(8):4945-55, 1993; Gabuzda, D. H., et al., J. AIDS 7(9):908-15, 1994).
As Vif is required by nonpermissive but not permissive cells for HIV-1 replication two possibilities exist. In permissive cells, there may be a Vif cellular homologue that can replace Vif function in the virus-producing cells; alternatively, there may be an inhibitor(s) of viral replication in nonpermissive cells that requires Vif to counteract its effect. (Trono, D., Cell 82:189-192, 1995). Recently, it was proposed that Vif protein is required to counteract an unknown endogenous inhibitor(s) in the virus-producing cells. (Madani, N., & Kabat, D., J. Virol. 72:10251-10255, 1998; Simon, J. H., et al., Nat. Med. 4:1397-1400, 1998). HIV-1 Vif can complement the function of HIV-1 Vif and SIVAGM Vif in human nonpermissive cells, whereas it cannot complement the function of HIV-1 and SIVAGM Vif in simian cells. SIVAGM Vif, however, can complement the function of HIV-1 Vif and SIVAGM Vif in simian cells but not the function of HIV-1 and SIVAGM Vif in human cells, indicating that a cellular cofactor(s) is involved in the action of Vif protein. (Simon, J. H., et al., EMBO J. 17:1259-1267, 1998). Conversely, since a Vif mutant (Vif from HIV-1FF12) can inhibit wild-type HIV-1 replication in permissive cells, a Vif homologue in the permissive cells may exist. (D'Aloja, P., et al., J. Virol. 72:4308-4319, 1998).
It has been proposed that Vif functions in virus-producing cells or cell-free virions and affects viral assembly. (Blanc, D., et al., Virology 193:186-192, 1993; Gabuzda, D. H., et al., J. Virol. 66:6489-6495, 1992; von Schwedler, U., et al., J. Virol. 67:4945-4955, 1993). Defects of the vif gene do not have detectable effects on viral transcription and translation or on virion production. HIV-1 variants with a defective vif gene are able to bind and penetrate target cells but are not able to complete intracellular reverse transcription and endogenous reverse transcription (ERT) in cell-free virions. (Courcoul, M., et al., J. Virol. 69:2068-2074, 1995; Goncalves, J., et al., J. Virol. 70:8701-8709, 1996; Sova, P., & Volsky, D. J., J. Virol. 67:6322-6326, 1993; von Schwedler, U., et al., J. Virol. 67:4945-4955, 1993). When ERT is driven by the addition of deoxyribonucleoside triphophates (DNTP) at high concentrations, certain levels of plus-strand viral DNA can be completed. Moreover, when vif− viruses, generated from nonpermissive cells and harboring larger quantities of viral DNA generated by ERT, are allowed to infect permissive cells, they can partially bypass the block at intracellular reverse transcription through which vif− viruses without deoxynucleoside triphosphate treatment can not pass. Consequently, viral infectivity can be partially rescued from the vif− phenotype. (Dornadula, G., et al., J. Virol. 74:2594-2602, 2000).
The expression of viral components, including viral proteins and nucleic acids, is not altered in the virions produced from nonpermissive cells. (Fouchier, R. A., et al., J. Virol. 70:8263-8269, 1996; Gabuzda, D. H., et al., J. Virol. 66:6489-6495, 1992; von Schwedler, U., et al., J. Virol. 67:4945-4955, 1993). Deletion of the vif gene, however, results in alterations of virion morphology. (Borman, A. M., et al., J. Virol. 69:2058-2067, 1995; Bouyac, M., et al., J. Virol 71:2473-2477, 1997; Hoglund, S., et al., Virology 201:349-355, 1994). The quantity of Vif protein in the HIV-1 virions generated from chronically infected cells is approximately 7 to 28 molecules per virion. (Camaur, D., & Trono, D., J. Virol. 70:6106-6111, 1996; Fouchier, R. A., et al., J. Virol. 70:8263-8269, 1996; Simon, J. H., et al., Virology 248:182-187, 1998). As the virion-associated Vif proteins do not depend on the expression of viral components and the amount of Vif in the virus-producing cells, it seems that Vif proteins are not specifically incorporated into the virions. (Camaur, D., & Trono, D., J. Virol 70:6106-6111, 1996; Simon, J. H., et al., Virology 248:182-187, 1998).
Although, it seems that Vif is not specifically incorporated into virions, Vif is able to bind to the NCp7 domain of p55 Gag precursors through its positively charged amino-acid enriched C-terminus. (Bouyac, M., et al., J. Virol. 71:9358-9365, 1997; Huvent, I., et al., J. Gen. Virol. 79:1069-1081, 1998). Vif protein is found to co-localize with Gag precursors in the cytoplasm of HIV-1-infected cells. (Simon, J. H., et al., J. Virol. 71:5259-5267, 1997). The molar ration of Vif to Gag precursors in infected cells is 1:1.7, suggesting that Vif plays a structural rather than a regulatory role in virus-producing cells. (Goncalves, J., et al., J. Virol. 68:704-712, 1994; Simon, J. H., et al., Virology 248:182-187, 1998).
Vif has been shown to be an RNA-binding protein and an integral component of a messenger ribonucleoprotein (mRNP) complex of viral RNA in the cytoplasm of HIV-1-infected cells. The expression of Vif in infected cells is quite high, and the majority of Vif in virus-producing cells is in the cytoplasmic fraction; some is associated with the cellular membrane. The Vif protein in this mRNP complex may protect viral RNA from various endogenous inhibitors and could mediate viral RNA engagement with HIV-1 Gag precursors and thus could be involved in genomic RNA folding and packaging. As such, the interaction between Vif and HIV-1 RNA plays an important role in the late events of the HIV-1 life cycle. Given the Vif protein's direct or indirect involvement in the viral assembly process, it is an ideal target for anti-HIV-1 therapeutics.
Many HIV-1 proteins, including Gag, protease, reverse transcriptase, integrase, glycoprotein 41(gp41), Tat, Rev, Vpr, and Nef, have been shown to form dimers or multimers in vitro and in vivo. The formation of dimers or multimers has been demonstrated to be important for their functions in the lentiviral life-cycle. (Frankel, A. D. & Young, J. A., Ann. Rev. Biochem. 67:1-25, 1998; Vaishnav, Y. N. & Wong-Staal, F., Annu Rev Biochem 60:577-630, 1991; Zhao, L. J., et al., J Biol Chem 269(51):32131-7, 1994; Liu, L., et al., J. Virol. 74:5310-5319, 2000). The present invention provides evidence that Vif protein possesses a strong tendency to self-associate and that multimerization of Vif proteins is important for Vif function in the viral life-cycle. The present invention is directed to a method of treating HIV-1 exposed or infected individuals by administering a composition that inhibits or prevents the replicative and other essential functions of Vif by binding to, or otherwise associating with, the multimerization domain of Vif, thereby preventing multimerization of Vif and, consequently, HIV-1 replication.